Fibrin glue from single-donation autologous plasmapheresis B. CASALI,F. RODEGHIERO, A. TOSE-ITO,B. PALMIERI, R. IMMOVILLI, C. GHEDINI,AND P. RIVASI
is described for preparing fibrin glue from fibrinogen obtained by double cryoprecipitation of plasma collected by piasmapheresis. Plasma (average volume, 528.8 f 86 mL), collected from 12 plasmapheresis donors w a s cryoprecipitated twice. When the cryoprecipitated pellet w a s resuspended in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL), was 78.4 k 18.3 mg per mL. This is comparable to suitable for clinical use, and offers greater safety a t a A method
1992;32:641-643.
Abbreviations: F Vlll = factor VIII; SDS = sodium dodecyl sulfate.
FIBRINGLUE
IS A biologic sealant obtained
from hu-
man fibrinogen mixed with bovine thrombin and calcium chloride. Fibrin glue has been employed in surgery of parenchymal organs such as liver,' lung,2 and ~ p l e e n . ~ Occasionally, its use has been reported also in nerve and vessel repail.4~~ and skin grafte6 In these instances, this biologic sealant has been found to be effective in promoting optimal hemostasis and wound repair.6 Many methods have been described for concentrating fibrinogen from plasma; the simplest is cryoprecipitation. A limitation of this procedure is that the quantity of fibrinogen recovered from a single donation is usually insufficient for clinical use, which makes autologous plasma donation troublesome. We report a simple method for the preparation of fibrin glue from a single plasmapheresis donation.
Materials and Methods
Fibrin glue preparation Plasma (8 m u g ) was collected from 12 healthy donors at a collection rate of 100 mL per minute and using plasmapheresis equipment (Autoapheresis, Haemoscience Laboratories, Santa Ana, CA) with a double-bag system. This system collects the plasma with a Plasmacell-C membrane device. and returns the residual red cell concentrate to the donor. The collecting system consists of two connected 1000-mL ethyl-vinylacetate bags, designed by one of us (CB)and produced by Miramed (Mirandola, Italy). When the procedure was completed, 300 to 400 mL of saline was automatically infused. CPDA, at an 8-percent volume-pervolume anticoagulant ratio, was the anticoagulant.
Immediately after plasmapheresis, the bags of plasma were stored at -80°C for at least 12 hours and subsequently thawed at 4°C for 16 hours. After an initial centrifugation at loo0 X g for 15 minutes at 4"C, we froze the material and thawed it once more as described above, being careful not to disturb the precipitated pellet. This second cryoprecipitation was introduced to increase the fibrinogen yield according to the suggestion of Wan et al.' The time between the first and s a n d thaw was about 8 hours, and during the freeze-thaw process, the units were kept upright, so as to maintain the pellet. After the second centrifugation, we drained all the supernatant plasma into an integral satellite bag and stored the cry+ precipitate at -30°C as a dry button that required thawing before resuspension. The operator time required to complete this procedure is about 1 hour. We obtained the fibrin glue by resuspending the fibrinogenrich cryoprecipitate in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL) and mixing an equal volume of that mixture and bovine thrombin (500 NIH units/mL, Topostasin, Roche, Milan, Italy) using a Y set (Miramed). The cryoprecipitate was resuspended in 0.5 to 2 mL of saline to produce. a quantity of fibrin glue suitable for major surgery; a smaller volume (1 mL) may be used for minor surgical procedures (e.g., tooth extractions).
Biochemical analysis and tension strength measurement We carried out protein electrophoresis using sodium dodecyl sulfate (SDS)4-percent and a polyacrylamide gel slab in a 15well vertical electrophoresis apparatus (Protean, BioRad, Richmond, CA). Sample buffer was SDS 2-percent, Tris-HC10.125 M (0.125 mom), sodium iodoacetate 0.004 M (0.004 mow). We used high-molecular-weight protein standards (Phannacia LKB. Milan, Italy) for molecular weight calibration. Fibrinogen content was determined by the method of C l a m , based on clottability with thrombh8 Fibronectin content was measured with an enzyme-linked immunosorbent assay kit (Behringwerke, Marburg, Germany). Factor XI11 (FXIII) subunit A and subunit B were measured using Laurell electroimmunoassay, according to a previously published m ~ t h o d .Transamidase ~ activity of factor X m was determined by the dansylcadaverine-casein incorporation assay.lo We measured plasminogen by Laurell electroimmunoassay, using purified plasminogen as a standard (provided
From the Ccntro Trasfusionale, Ospedale S. Maria, Reggio Emilia, Italy; Centro Regionale per lo Studio delle Malatte Emorragiche e Trombotiche. Divisione di Ematologia, Ospedalc San Bortolo, Vicenza, Italy; Clinica Chirurgica, Policlinico di Modena, Modena, Italy; and Miramed, Mirandola. Modena. Italy. Received for publication September 25,1991;revision received January 31, 1992, and accepted February 11, 1992.
641
642
TRANSF'USION Vol. 32. No. 7-1992
CASALI ET AL.
by Dr. G. Delchin, Stago, Milan, Italy). We determined albumin levels by immunonephelometry (Perkin-Elmer, Monza, Italy) and measured tensile strength by the Gottlob mouse skin method." Briefly, four pieces of mouse skin measuring 1 cm2 were removed from the mouse back, washed with saline and connected, through a nylon wire, to a pulley. The two components of the fibrin glue (50 p L of bovine thrombin and 50 p L of cryoprecipitated fibrinogen) were layered evenly on each of the patches, which were immediately replaced on the mouse back. After 60 and 120 minutes, we measured the tension (g! cm2) required to peel off the skin patches by adding a standard weight to the pulley.
Table 1.
Biochemical composition of cryoprecipitate*
Fibrinogen (mglml) Factor Xlll transamidase activity (UlrnL) Factor Xlll subunit A (UlmL) Factor Xlll subunit B (UlmL) Plasminogen (pglml) Fibronectin (KglmL) Albumin (gldL)
78.4 f 18.0 15.8 2 6 15.3 2 4 16.3 2 5 113.7 f 44 9624.4 f 847.5 2.7 & 0.8
Mean f SD from 12 separate preparations;cryoprecipitatepellet was resuspended in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL).
Results The average volume of plasma collected by apheresis from 12 donors was 528 f 86 mL. The apheresis procedure required about 30 minutes. SDS-polyacrylamide gel electrophoresis of our cryoprecipitate and of a commercial fibrin glue (Tissucol, Immuno, Vienna, Austria) showed a protein band with an apparent molecular mass of 340,000 daltons, identifiable as fibrinogen (Fig. 1). An albumin band was also present. Table 1 presents the main biochemical findings. The fibrinogen concentration was 78.4 & 18.0 mg per mL (total amount 394.0 & 91.5 mg in the 5 mL of saline-resuspended fibrinogen). FXIII transamidase activity and subunit A and subunit B concentration were, respectively, 15.8 f 6, 15.3 & 4, and 16.3 & 5 U per mL when measured against a normal pooled plasma (assumed to contain 1 U/mL of FXIII). Plasminogen concentration was 113.7 & 44 pg per mL, fibronectin content was 9624.4 f 847.5 pg per mL, and the albumin level was
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human albumin, lane 1; purified fibrinogen commercial standard, lane 2; cryoprecipitate obtained by a double thawing and freezing method, lanes 3 and 4; fibrinogen from a commercial fibrin glue, lane 5: and arotein standard with molecular weieht (MWI markers. lane 6. I
.
Y
,
I
Table 2.
Tensile strength of fibrin glue*
Time after skin application (minutes)
Tensile strength (g/cm2)
60 120
117 15 130 f 31
Mean
2
SD, 7 preparations.
2.7 f 0.8 g per dL. The tensile strength measured in the mouse is reported in Table 2.
Discussion
This study shows that a fibrinogen concentrate suitable for fibrin glue can be obtained by a double cryoprecipitation from a single plasmapheresis donation. As shown in other studies,7.12the properties of fibrin glue are determined mainly by the presence of fibrinogen, FXIII and fibronectin. Fibrinogen content is related to the tensile strength of the fibrin glue, which is also increased by the FXIII-mediated cross-linking of fibrinogen. The quantity of fibrinogen recovered (78.4 f 18.3 mg/mL; total amount, 394.0 & 91.5 mg) is similar to that of commercial fibrin glue (70-100 mg/mL; Tissucol, Immuno). FXIII transamidase activity in the cryoprecipitateclosely correlates with both FXIII subunit A and B antigen, so that no appreciable denaturation of FXIII occurred. Fibrin glue obtained from pooled plasma12 and from the 5-mL commercial kit (Tissucol; data as reported by the manufacturer) show, respectively, 5.9 f 0.51 g per L and 10 to 45 mg of fibronectin, levels that are lower than those present in our product. The role of fibronectin in fibrin glue is controversial. Fibronectin has been found to improve wound healing by favoring fibroblast adhesion and tissue reepithelization,13J4 but it has also been shown to reduce fibroblast adhesion" and to decrease fibrin polymerization.16 Albumin may prevent fibrin gelation by increasing fibrin ~olubility.'~ The plasminogen concentration in our preparation is comparable to that in normal plasma'* and should not lead to fibrinolysis.
TRANSFUSION 1992-Vol. 32, No. 7
AUTOLOGOUS FIBRIN GLUE PREPARATION
The composition of the fibrin glue obtained by our method, as shown in Table 1, seems particularly favorable and is able to promote a tensile strength comparable to that of fibrin glue obtained from pooled plasma.19 In one study,’ the tensile strength of a fibrin glue containing 60 mg per dL of fibrinogen was 620 g per cm2; however, a direct comparison between the tensile strengths in that study and in ours is not possible because in that study, the animal model used is not reported. Other advantages of our method are the completely closed multi-bag system, rapid completion (within 48 hours) of the procedure, and the short time (< 40 minutes) spent by the donor. Moreover, the work required of the laboratory staff takes less than 1 hour. From December 1989 to January 1991, we used autologous fibrin glue prepared by this method in elective surgery, including thoracic, liver, and urologic surgery, in 47 patients. Autologous fibrin glue was effective in controlling diffuse parenchymal hemorrhage and bleeding foci after lung lobectomy or partial hepatectomy and for sealing urethrointestinal and ureterovesical anastomosis. There was no abscess formation, delayed hematoma, or allergic reaction. Although disease transmission by commercial fibrin glue preparations has not been proved in a retrospective study,20 and although fibrinogen obtained from pooled plasma is heat-treated, the risk of virus transmission has not been completely eliminated. Fibrin glue from single autologous plasmapheresis donation and even from single-donor cryoprecipitate may be safer than fibrin glue prepared by pooling several units. An additional reason for the use of autologous fibrin glue is the report of human immunodeficiency virus type 1 transmission by topical use of fibrin glue produced by using 2 allogeneic units of cryoprecipitate.21 In emergency surgery (e. g., repair of traumatic laceration of spleen or liver), fibrin glue from a single wellscreened donor may be advantageous. The cost of our preparation compares favorably with that of commercial preparations, being around $97, including the plasmapheresis kit. In conclusion, our study shows the feasibility of obtaining fibrin glue from cryoprecipitated fibrinogen recovered from single autologous or allogeneic plasmapheresis donations. This procedure is simple, safe, and widely applicable and abolishes the risk of transmission of blood-borne virus. References 1. Bergsland J, Kalmbach T, Balu D, Feldman MJ, Carvana JA, Gage AA. Fibrin seal-an alternative to suture repair in experimental pulmonary surgery. J Surg Res 1986;40:340-5. 2. Jakob H, Campbell CD, Stemberger A, Wriedt-Lubbe I, Blumel G. Replogie RL. Combined application of heterologous collagen and fibrin sealant for liver injuries. J Surg Res 1984;36:571-7.
643
3. Kram HB, Shumaker WC, Hino ST, Harley DP. Splenic salvage using biologic glue. Arch Surg 1984;1191309-11. 4. Ventura R, Tom G, Campari A, Giandomenico A, Peretti G. Experimental suture of the peripheral nerves with “fibrin glue”. Ital J Orthop Traumatol 1981;6:407-14. 5. Barton B, Moore EE, Pearce WH. Fibrin glue as a biologic vascular patch-a comparative study. J Surg Res 1986;40:510-3. 6. Tidrick RT, Warner ED. Fibrin furation of skin transplants. Surgery 1944,15:90-5. 7. Wan HL, Huang ST, Floyd DM. McGowan El, Feldman DS. Is the amount of fibrinogen in cryoprecipitate adquate for fibrin glue? Introducing an improved rccyclcd cryoprecipitate method (abstract). Transfusion 1989;29(Suppl):41S. 8. Von Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957;17:237-46. 9. Rodeghiero F, Morbin M, Barbui T. Subunit A of Factor XI11 regulates subunit B plasma concentration. Thromb Haemost
1981;46:621-2. 10. Rodeghiero F, Barbui T. Fibrin cross-linking in congenital Factor XI11 deficiency. J Clin Pathol 1980;33:434-7. 11. Gestring GF, Lcrner R. Autologous fibrinogen for tissue-adhesion, hemostasis and embolization. Vasc Surg 1983;17:294-304.
14. Burnouf-Radosevich M. Burnouf T, Huart JJ. Biochemical and
physical properties of soh.tntdetergent-tated fibrin glue. Vox Sang 1990;58:77-84. 13. Grinnell F, Feld MK. Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin. Cell
1979;17:117-29. 14. Igisu K. The role of fibronectin in the proccss of wound healing. Thromb Res 1986;44:455-65. 15. Bruhn HD. Pohl J. Growth regulation of fibroblasts by thrombin, factor XI11 and fibronectin. Klin Wochenschr 1981;59:145-6. 16. Niewiarowska J, Cierniewski CS. Inhibitory effect of fibronectin on the fibrin formation. Thromb Res 1982;27:611-8. 17. Galanakis DK, Lane BP, Simon SR. Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen. Biochemistry
1987;262389-400. 18. Collen D, Verstraete M. Molecular biology of human plasminogen. 11. Metabolism in physiological and some pathological conditions in man. Thromb Diath Haemorrh 1975;34:403-8. 19. Burnouf-Radosevich M, Dwal P, Burnouf T, et el. Lc colles biologique: composition prottque et qualit&de colles biologique europbnnes. Lyon Chir 1988;84:191-5. 20. Cain JE Jr. Dryer RF, Barton BR. Evaluation of dural closure techniques. Suture methods, fibrin adhesive sealant and cyanoacrylate polymer. Spine 1988;13:720-5. 21. Wilson SM, Pel1 P, Donegan EA. HIV-1 transmission following the use of cryoprecipitated fibrinogen as geVadhesive (abstract). Transfusion 1991;31(Suppl):SlS. Bruno Casali, MD, Assistant, Blood Transfusion Service, Santa Maria Hospital. Francesco Rodeghiero, MD, Director, Hemophilia and Thrombosis Centre, Department of Hematology, San Bortolo Hospital, Vicem, Italy. Albert0 Tosctto, MD, Assistant, Hemophilia and ’IhrombosisCentre, Department of Hematology, San Bortolo Hospital. Beniamino Palmieri, MD, Associate Professor, Department of Surgery, University of Modena, Modena. Italy. Roberta Immovilli, Biol. Sc., Assistant. Blood Transfusion Service, Santa Maria Hospital. Claudia Ghedini. Biol. Sc., Technologist, Miramed, Mirandola. Modena, Italy. Paolo Rivasi, MD, Director, Blood Transfusion Service,Santa Maria Hospital, viale Risorgimento 80, Emilia Romagna 42100,Reggio Emilia, Italy. [Reprint requests].
is described for preparing fibrin glue from fibrinogen obtained by double cryoprecipitation of plasma collected by piasmapheresis. Plasma (average volume, 528.8 f 86 mL), collected from 12 plasmapheresis donors w a s cryoprecipitated twice. When the cryoprecipitated pellet w a s resuspended in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL), was 78.4 k 18.3 mg per mL. This is comparable to suitable for clinical use, and offers greater safety a t a A method
1992;32:641-643.
Abbreviations: F Vlll = factor VIII; SDS = sodium dodecyl sulfate.
FIBRINGLUE
IS A biologic sealant obtained
from hu-
man fibrinogen mixed with bovine thrombin and calcium chloride. Fibrin glue has been employed in surgery of parenchymal organs such as liver,' lung,2 and ~ p l e e n . ~ Occasionally, its use has been reported also in nerve and vessel repail.4~~ and skin grafte6 In these instances, this biologic sealant has been found to be effective in promoting optimal hemostasis and wound repair.6 Many methods have been described for concentrating fibrinogen from plasma; the simplest is cryoprecipitation. A limitation of this procedure is that the quantity of fibrinogen recovered from a single donation is usually insufficient for clinical use, which makes autologous plasma donation troublesome. We report a simple method for the preparation of fibrin glue from a single plasmapheresis donation.
Materials and Methods
Fibrin glue preparation Plasma (8 m u g ) was collected from 12 healthy donors at a collection rate of 100 mL per minute and using plasmapheresis equipment (Autoapheresis, Haemoscience Laboratories, Santa Ana, CA) with a double-bag system. This system collects the plasma with a Plasmacell-C membrane device. and returns the residual red cell concentrate to the donor. The collecting system consists of two connected 1000-mL ethyl-vinylacetate bags, designed by one of us (CB)and produced by Miramed (Mirandola, Italy). When the procedure was completed, 300 to 400 mL of saline was automatically infused. CPDA, at an 8-percent volume-pervolume anticoagulant ratio, was the anticoagulant.
Immediately after plasmapheresis, the bags of plasma were stored at -80°C for at least 12 hours and subsequently thawed at 4°C for 16 hours. After an initial centrifugation at loo0 X g for 15 minutes at 4"C, we froze the material and thawed it once more as described above, being careful not to disturb the precipitated pellet. This second cryoprecipitation was introduced to increase the fibrinogen yield according to the suggestion of Wan et al.' The time between the first and s a n d thaw was about 8 hours, and during the freeze-thaw process, the units were kept upright, so as to maintain the pellet. After the second centrifugation, we drained all the supernatant plasma into an integral satellite bag and stored the cry+ precipitate at -30°C as a dry button that required thawing before resuspension. The operator time required to complete this procedure is about 1 hour. We obtained the fibrin glue by resuspending the fibrinogenrich cryoprecipitate in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL) and mixing an equal volume of that mixture and bovine thrombin (500 NIH units/mL, Topostasin, Roche, Milan, Italy) using a Y set (Miramed). The cryoprecipitate was resuspended in 0.5 to 2 mL of saline to produce. a quantity of fibrin glue suitable for major surgery; a smaller volume (1 mL) may be used for minor surgical procedures (e.g., tooth extractions).
Biochemical analysis and tension strength measurement We carried out protein electrophoresis using sodium dodecyl sulfate (SDS)4-percent and a polyacrylamide gel slab in a 15well vertical electrophoresis apparatus (Protean, BioRad, Richmond, CA). Sample buffer was SDS 2-percent, Tris-HC10.125 M (0.125 mom), sodium iodoacetate 0.004 M (0.004 mow). We used high-molecular-weight protein standards (Phannacia LKB. Milan, Italy) for molecular weight calibration. Fibrinogen content was determined by the method of C l a m , based on clottability with thrombh8 Fibronectin content was measured with an enzyme-linked immunosorbent assay kit (Behringwerke, Marburg, Germany). Factor XI11 (FXIII) subunit A and subunit B were measured using Laurell electroimmunoassay, according to a previously published m ~ t h o d .Transamidase ~ activity of factor X m was determined by the dansylcadaverine-casein incorporation assay.lo We measured plasminogen by Laurell electroimmunoassay, using purified plasminogen as a standard (provided
From the Ccntro Trasfusionale, Ospedale S. Maria, Reggio Emilia, Italy; Centro Regionale per lo Studio delle Malatte Emorragiche e Trombotiche. Divisione di Ematologia, Ospedalc San Bortolo, Vicenza, Italy; Clinica Chirurgica, Policlinico di Modena, Modena, Italy; and Miramed, Mirandola. Modena. Italy. Received for publication September 25,1991;revision received January 31, 1992, and accepted February 11, 1992.
641
642
TRANSF'USION Vol. 32. No. 7-1992
CASALI ET AL.
by Dr. G. Delchin, Stago, Milan, Italy). We determined albumin levels by immunonephelometry (Perkin-Elmer, Monza, Italy) and measured tensile strength by the Gottlob mouse skin method." Briefly, four pieces of mouse skin measuring 1 cm2 were removed from the mouse back, washed with saline and connected, through a nylon wire, to a pulley. The two components of the fibrin glue (50 p L of bovine thrombin and 50 p L of cryoprecipitated fibrinogen) were layered evenly on each of the patches, which were immediately replaced on the mouse back. After 60 and 120 minutes, we measured the tension (g! cm2) required to peel off the skin patches by adding a standard weight to the pulley.
Table 1.
Biochemical composition of cryoprecipitate*
Fibrinogen (mglml) Factor Xlll transamidase activity (UlrnL) Factor Xlll subunit A (UlmL) Factor Xlll subunit B (UlmL) Plasminogen (pglml) Fibronectin (KglmL) Albumin (gldL)
78.4 f 18.0 15.8 2 6 15.3 2 4 16.3 2 5 113.7 f 44 9624.4 f 847.5 2.7 & 0.8
Mean f SD from 12 separate preparations;cryoprecipitatepellet was resuspended in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL).
Results The average volume of plasma collected by apheresis from 12 donors was 528 f 86 mL. The apheresis procedure required about 30 minutes. SDS-polyacrylamide gel electrophoresis of our cryoprecipitate and of a commercial fibrin glue (Tissucol, Immuno, Vienna, Austria) showed a protein band with an apparent molecular mass of 340,000 daltons, identifiable as fibrinogen (Fig. 1). An albumin band was also present. Table 1 presents the main biochemical findings. The fibrinogen concentration was 78.4 & 18.0 mg per mL (total amount 394.0 & 91.5 mg in the 5 mL of saline-resuspended fibrinogen). FXIII transamidase activity and subunit A and subunit B concentration were, respectively, 15.8 f 6, 15.3 & 4, and 16.3 & 5 U per mL when measured against a normal pooled plasma (assumed to contain 1 U/mL of FXIII). Plasminogen concentration was 113.7 & 44 pg per mL, fibronectin content was 9624.4 f 847.5 pg per mL, and the albumin level was
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human albumin, lane 1; purified fibrinogen commercial standard, lane 2; cryoprecipitate obtained by a double thawing and freezing method, lanes 3 and 4; fibrinogen from a commercial fibrin glue, lane 5: and arotein standard with molecular weieht (MWI markers. lane 6. I
.
Y
,
I
Table 2.
Tensile strength of fibrin glue*
Time after skin application (minutes)
Tensile strength (g/cm2)
60 120
117 15 130 f 31
Mean
2
SD, 7 preparations.
2.7 f 0.8 g per dL. The tensile strength measured in the mouse is reported in Table 2.
Discussion
This study shows that a fibrinogen concentrate suitable for fibrin glue can be obtained by a double cryoprecipitation from a single plasmapheresis donation. As shown in other studies,7.12the properties of fibrin glue are determined mainly by the presence of fibrinogen, FXIII and fibronectin. Fibrinogen content is related to the tensile strength of the fibrin glue, which is also increased by the FXIII-mediated cross-linking of fibrinogen. The quantity of fibrinogen recovered (78.4 f 18.3 mg/mL; total amount, 394.0 & 91.5 mg) is similar to that of commercial fibrin glue (70-100 mg/mL; Tissucol, Immuno). FXIII transamidase activity in the cryoprecipitateclosely correlates with both FXIII subunit A and B antigen, so that no appreciable denaturation of FXIII occurred. Fibrin glue obtained from pooled plasma12 and from the 5-mL commercial kit (Tissucol; data as reported by the manufacturer) show, respectively, 5.9 f 0.51 g per L and 10 to 45 mg of fibronectin, levels that are lower than those present in our product. The role of fibronectin in fibrin glue is controversial. Fibronectin has been found to improve wound healing by favoring fibroblast adhesion and tissue reepithelization,13J4 but it has also been shown to reduce fibroblast adhesion" and to decrease fibrin polymerization.16 Albumin may prevent fibrin gelation by increasing fibrin ~olubility.'~ The plasminogen concentration in our preparation is comparable to that in normal plasma'* and should not lead to fibrinolysis.
TRANSFUSION 1992-Vol. 32, No. 7
AUTOLOGOUS FIBRIN GLUE PREPARATION
The composition of the fibrin glue obtained by our method, as shown in Table 1, seems particularly favorable and is able to promote a tensile strength comparable to that of fibrin glue obtained from pooled plasma.19 In one study,’ the tensile strength of a fibrin glue containing 60 mg per dL of fibrinogen was 620 g per cm2; however, a direct comparison between the tensile strengths in that study and in ours is not possible because in that study, the animal model used is not reported. Other advantages of our method are the completely closed multi-bag system, rapid completion (within 48 hours) of the procedure, and the short time (< 40 minutes) spent by the donor. Moreover, the work required of the laboratory staff takes less than 1 hour. From December 1989 to January 1991, we used autologous fibrin glue prepared by this method in elective surgery, including thoracic, liver, and urologic surgery, in 47 patients. Autologous fibrin glue was effective in controlling diffuse parenchymal hemorrhage and bleeding foci after lung lobectomy or partial hepatectomy and for sealing urethrointestinal and ureterovesical anastomosis. There was no abscess formation, delayed hematoma, or allergic reaction. Although disease transmission by commercial fibrin glue preparations has not been proved in a retrospective study,20 and although fibrinogen obtained from pooled plasma is heat-treated, the risk of virus transmission has not been completely eliminated. Fibrin glue from single autologous plasmapheresis donation and even from single-donor cryoprecipitate may be safer than fibrin glue prepared by pooling several units. An additional reason for the use of autologous fibrin glue is the report of human immunodeficiency virus type 1 transmission by topical use of fibrin glue produced by using 2 allogeneic units of cryoprecipitate.21 In emergency surgery (e. g., repair of traumatic laceration of spleen or liver), fibrin glue from a single wellscreened donor may be advantageous. The cost of our preparation compares favorably with that of commercial preparations, being around $97, including the plasmapheresis kit. In conclusion, our study shows the feasibility of obtaining fibrin glue from cryoprecipitated fibrinogen recovered from single autologous or allogeneic plasmapheresis donations. This procedure is simple, safe, and widely applicable and abolishes the risk of transmission of blood-borne virus. References 1. Bergsland J, Kalmbach T, Balu D, Feldman MJ, Carvana JA, Gage AA. Fibrin seal-an alternative to suture repair in experimental pulmonary surgery. J Surg Res 1986;40:340-5. 2. Jakob H, Campbell CD, Stemberger A, Wriedt-Lubbe I, Blumel G. Replogie RL. Combined application of heterologous collagen and fibrin sealant for liver injuries. J Surg Res 1984;36:571-7.
643
3. Kram HB, Shumaker WC, Hino ST, Harley DP. Splenic salvage using biologic glue. Arch Surg 1984;1191309-11. 4. Ventura R, Tom G, Campari A, Giandomenico A, Peretti G. Experimental suture of the peripheral nerves with “fibrin glue”. Ital J Orthop Traumatol 1981;6:407-14. 5. Barton B, Moore EE, Pearce WH. Fibrin glue as a biologic vascular patch-a comparative study. J Surg Res 1986;40:510-3. 6. Tidrick RT, Warner ED. Fibrin furation of skin transplants. Surgery 1944,15:90-5. 7. Wan HL, Huang ST, Floyd DM. McGowan El, Feldman DS. Is the amount of fibrinogen in cryoprecipitate adquate for fibrin glue? Introducing an improved rccyclcd cryoprecipitate method (abstract). Transfusion 1989;29(Suppl):41S. 8. Von Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957;17:237-46. 9. Rodeghiero F, Morbin M, Barbui T. Subunit A of Factor XI11 regulates subunit B plasma concentration. Thromb Haemost
1981;46:621-2. 10. Rodeghiero F, Barbui T. Fibrin cross-linking in congenital Factor XI11 deficiency. J Clin Pathol 1980;33:434-7. 11. Gestring GF, Lcrner R. Autologous fibrinogen for tissue-adhesion, hemostasis and embolization. Vasc Surg 1983;17:294-304.
14. Burnouf-Radosevich M. Burnouf T, Huart JJ. Biochemical and
physical properties of soh.tntdetergent-tated fibrin glue. Vox Sang 1990;58:77-84. 13. Grinnell F, Feld MK. Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin. Cell
1979;17:117-29. 14. Igisu K. The role of fibronectin in the proccss of wound healing. Thromb Res 1986;44:455-65. 15. Bruhn HD. Pohl J. Growth regulation of fibroblasts by thrombin, factor XI11 and fibronectin. Klin Wochenschr 1981;59:145-6. 16. Niewiarowska J, Cierniewski CS. Inhibitory effect of fibronectin on the fibrin formation. Thromb Res 1982;27:611-8. 17. Galanakis DK, Lane BP, Simon SR. Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen. Biochemistry
1987;262389-400. 18. Collen D, Verstraete M. Molecular biology of human plasminogen. 11. Metabolism in physiological and some pathological conditions in man. Thromb Diath Haemorrh 1975;34:403-8. 19. Burnouf-Radosevich M, Dwal P, Burnouf T, et el. Lc colles biologique: composition prottque et qualit&de colles biologique europbnnes. Lyon Chir 1988;84:191-5. 20. Cain JE Jr. Dryer RF, Barton BR. Evaluation of dural closure techniques. Suture methods, fibrin adhesive sealant and cyanoacrylate polymer. Spine 1988;13:720-5. 21. Wilson SM, Pel1 P, Donegan EA. HIV-1 transmission following the use of cryoprecipitated fibrinogen as geVadhesive (abstract). Transfusion 1991;31(Suppl):SlS. Bruno Casali, MD, Assistant, Blood Transfusion Service, Santa Maria Hospital. Francesco Rodeghiero, MD, Director, Hemophilia and Thrombosis Centre, Department of Hematology, San Bortolo Hospital, Vicem, Italy. Albert0 Tosctto, MD, Assistant, Hemophilia and ’IhrombosisCentre, Department of Hematology, San Bortolo Hospital. Beniamino Palmieri, MD, Associate Professor, Department of Surgery, University of Modena, Modena. Italy. Roberta Immovilli, Biol. Sc., Assistant. Blood Transfusion Service, Santa Maria Hospital. Claudia Ghedini. Biol. Sc., Technologist, Miramed, Mirandola. Modena, Italy. Paolo Rivasi, MD, Director, Blood Transfusion Service,Santa Maria Hospital, viale Risorgimento 80, Emilia Romagna 42100,Reggio Emilia, Italy. [Reprint requests].
